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A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Rachael E. Workman, Teja Pammi, Binh T. K. Nguyen, Leonardo W. Graeff, Erika Smith, Suzanne M. Sebald, Marie J. Stoltzfus, View ORCID ProfileJoshua W. Modell
doi: https://doi.org/10.1101/2020.05.21.102756
Rachael E. Workman
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Teja Pammi
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Binh T. K. Nguyen
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Leonardo W. Graeff
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Erika Smith
2Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Suzanne M. Sebald
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Marie J. Stoltzfus
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Joshua W. Modell
1Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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  • ORCID record for Joshua W. Modell
  • For correspondence: jmodell1@jhmi.edu
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SUMMARY

CRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9 serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted May 21, 2020.
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A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression
Rachael E. Workman, Teja Pammi, Binh T. K. Nguyen, Leonardo W. Graeff, Erika Smith, Suzanne M. Sebald, Marie J. Stoltzfus, Joshua W. Modell
bioRxiv 2020.05.21.102756; doi: https://doi.org/10.1101/2020.05.21.102756
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A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression
Rachael E. Workman, Teja Pammi, Binh T. K. Nguyen, Leonardo W. Graeff, Erika Smith, Suzanne M. Sebald, Marie J. Stoltzfus, Joshua W. Modell
bioRxiv 2020.05.21.102756; doi: https://doi.org/10.1101/2020.05.21.102756

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